Our broad aim is to elucidate how eukaryotic genomes are structured and how they work. We will focus on the role of heterochromatin. Normal development of animals, plants and fungi relies on chromatin features including methylation of DNA and histone H3K9 in constitutive heterochromatin, and methylation of histone H3K27 (H3K27me) in facultative heterochromatin. We have shown that the filamentous fungus Neurospora crassa is an extraordinarily favorable genetic/molecular system to elucidate the basic workings of both forms of heterochromatin. The project will involve genetic and molecular dissection of the interconnected roles of chromatin features implicated in heterochromatin including DNA signals, chromatin modifications, histone turnover, nucleosome organization, nuclear organization, and other factors. We have shown that a conserved protein complex (PRC2) is responsible for H3K27me in facultative heterochromatin and that this mark is repressive as in higher organisms. Importantly, H3K27me is not essential for viability of Neurospora, allowing for studies that would be difficult or impossible in higher organisms. A major objective of our study is to understand the mechanism of H3K27me-mediated transcriptional repression. We built a forward genetic scheme to identify genes required for H3K27me-mediated silencing and this has already identified interesting, unanticipated chromatin modifiers. We will both scale up our selection to identify more mutants and will characterize the factors already identified, testing if they act up- or down-stream of H3K27me and determining how they affect gene expression, nucleosome positioning, epigenetic modifications, and other features of chromatin. This will give insight into repression by H3K27me. In addition, we will focus on a tryptophan- inducible H3K27me-marked locus, kyn-1, for a controlled and in depth dissection of H3K27me regulation. We will also take several complementary approaches to elucidate what controls the genomic placement of this epigenetic mark. Our recent studies defined telomere-dependent (TD) and telomere-independent (TI) H3K27me and revealed that telomere repeats are capable of inducing H3K27me. We will investigate the underlying mechanism of TD H3K27me, for example by testing the possible roles of structural features (e.g. G- quadruplex DNA), telomere-associated proteins, and nuclear organization (e.g. placement at nuclear periphery). Use of gene knockouts and our LexA tethering system will allow us to test both necessity and sufficiency of candidate features. We will also experimentally dissect TI domains, which may identify ?PRE?-like elements and DNA binding factors involved in recruitment of H3K27me machinery. Finally, we will test how the broader chromatin environment controls H3K27me, following up our leads that suggest constitutive heterochromatin, H3K36me, H3K56ac, nucleosome turnover, and transcription all influence H3K27me distribution. We are optimistic that our findings on the control and function of heterochromatin in Neurospora will elucidate fundamental processes that also operate in higher eukaryotes.